This proposal is designed to investigate the structure of the processed influenza viral antigenic moieties generated in infected cells which are recognized by human CD4+ T lymphocytes and to characterize the intracellular pathways by which viral proteins are processed and presented to human T lymphocytes. The proposed studies are an extension of our ongoing work on the characterization of the human T lymphocyte response to viral polypeptides. Our experimental approach is to first characterize the naturally processed influenza hemagglutinin and nucleocapsid peptides bound to HLA DRwll by chromatographic and mass spectroscopic means. In related studies, the impact of virus infection on the spectrum of self peptide bound to MHC class II molecules in infected cells will be assessed as well as the effect of viral antigen form, e.g. isolated viral polypeptides or de novo expressed viral protein, on the structure of the naturally processed peptides generated in antigen presenting cells. Efforts will be made to generate reagents which can tract the formation of peptide MHC complexes in the infected cell and to evaluate the effect of aminoacids outside of an antigenic epitope on the processing of the viral polypeptides and the formation of the antigenic peptide. In related studies, we will examine the pathways of viral glycoprotein targeting to the lysosomal antigen processing compartment and the mechanism by which cytosolic viral proteins are processed and presented in association with human MHC class II molecule. The experiments outlined in this proposal will provide new information on the structure of the naturally processed viral antigenic moieties recognized human T lymphocytes, and the mechanism by which they are generated in virus infected cells. Such information will be of immediate importance in viral vaccine design and of long-term significance in understanding immune function during infection.